Molecular And Serological Characterization Of Avian Influenza Viruses In Domestic And Wild Birds

Material type: BookPublisher: 2012Subject(s): Department of Microbiology | Phd. thesisDDC classification: 1536,T Dissertation note: Avian Influenza virus (AIV) has been recognized one of the most important pathogen in poultry industry. AIVs play an important role in the pandemic spread that could cause high morbidity and mortality in human beings. A total of 1500 tracheal and cloacael swabs were collected from the seven live bird poultry markets of Lahore Pakistan for surveillance of AIV. The samples were processed for virus isolation in chicken embryos. The isolates were processed for HA test and AIV Antigen Rapid Test Kit to differentiate Newcastle disease virus and Avian influenza virus. Only four samples were positive for Avian influenza H9N2 subtype and 17 were positive for Newcastle disease virus. Four HA virus suspension of AIV showed high titers of anti AIV H9N2-HI antibody titer in chicken as well as rabbit serum, raised using Ottoman Pharma AIV-H9N2 (Oil based) vaccine.
The isolates of AIV H9N2 were confirmed using laboratory optimized RT-PCR, mRT-PCR and LAMP tests. All isolates were sequenced and analyzed to develop phylogenetic relationship. Two isolates A/Chicken/Pakistan/Micro-1/2009 and A/ chicken/ Pakistan/ Micro-2/ 2009 showed 99.1% nucleotide homology with each other and 95- 99% homology with the other Pakistani isolates, 95.1% homology with A/Chicken/Iran/B102/2005. The nucleotide sequence of "A/Duck/Pakistan/Micro-3/2009" showed 98.8% homology with "A/chicken/Pakistan/micro-4/2009", 98-98.7% homology with other Pakistani Isolates and 95.8% homology with "A/Chicken/Iran/B102/2005". The nucleotide sequence of "A/Chicken/Pakistan/Micro-4/2009" showed 99.6% homology with "A/duck/Pakistan/micro-3/2009", 95-96% homology with other Pakistani isolates and 94.3% homology with "A/Chicken/Iran/TH lBM863/2007".
Three out of four isolates had PARSSRGL cleavage sites and one isolate A/chicken/Pakistan/micro-1/2009 had PAKSSRGL cleavage site. The four isolates of the study contained a 226-Leu and 228-Gly at receptor binding sites. Substitution of Glutamine (Q) into Leucine (L) at 226 receptor binding site in HA glycoprotein increased the binding specificity for Sialic acid ? 2, 6 Galactose linkage of human receptor. The HA gene of the live poultry market isolates had 7 predicted glycosylation sites at 29-32, 105-108, 141-144, 298-301, 305-308, 492-495 and 551-554, positions. The NA gene of the live poultry market isolates had 8 predicted glycosylation sites at 44-46, 61-63, 69-71, 146-148, 200-203, 234-237 and 402-403, positions. Predicted glycosylation sites affected the structure and stability of NA protein.
It is concluded that continuous epidemiological and virological surveillance of live bird poultry markets may help scientists to develop an effective control and preventive measures for AI viruses.

Avian Influenza virus (AIV) has been recognized one of the most important pathogen in poultry industry. AIVs play an important role in the pandemic spread that could cause high morbidity and mortality in human beings. A total of 1500 tracheal and cloacael swabs were collected from the seven live bird poultry markets of Lahore Pakistan for surveillance of AIV. The samples were processed for virus isolation in chicken embryos. The isolates were processed for HA test and AIV Antigen Rapid Test Kit to differentiate Newcastle disease virus and Avian influenza virus. Only four samples were positive for Avian influenza H9N2 subtype and 17 were positive for Newcastle disease virus. Four HA virus suspension of AIV showed high titers of anti AIV H9N2-HI antibody titer in chicken as well as rabbit serum, raised using Ottoman Pharma AIV-H9N2 (Oil based) vaccine.

The isolates of AIV H9N2 were confirmed using laboratory optimized RT-PCR, mRT-PCR and LAMP tests. All isolates were sequenced and analyzed to develop phylogenetic relationship. Two isolates A/Chicken/Pakistan/Micro-1/2009 and A/ chicken/ Pakistan/ Micro-2/ 2009 showed 99.1% nucleotide homology with each other and 95- 99% homology with the other Pakistani isolates, 95.1% homology with A/Chicken/Iran/B102/2005. The nucleotide sequence of "A/Duck/Pakistan/Micro-3/2009" showed 98.8% homology with "A/chicken/Pakistan/micro-4/2009", 98-98.7% homology with other Pakistani Isolates and 95.8% homology with "A/Chicken/Iran/B102/2005". The nucleotide sequence of "A/Chicken/Pakistan/Micro-4/2009" showed 99.6% homology with "A/duck/Pakistan/micro-3/2009", 95-96% homology with other Pakistani isolates and 94.3% homology with "A/Chicken/Iran/TH lBM863/2007".

Three out of four isolates had PARSSRGL cleavage sites and one isolate A/chicken/Pakistan/micro-1/2009 had PAKSSRGL cleavage site. The four isolates of the study contained a 226-Leu and 228-Gly at receptor binding sites. Substitution of Glutamine (Q) into Leucine (L) at 226 receptor binding site in HA glycoprotein increased the binding specificity for Sialic acid ? 2, 6 Galactose linkage of human receptor. The HA gene of the live poultry market isolates had 7 predicted glycosylation sites at 29-32, 105-108, 141-144, 298-301, 305-308, 492-495 and 551-554, positions. The NA gene of the live poultry market isolates had 8 predicted glycosylation sites at 44-46, 61-63, 69-71, 146-148, 200-203, 234-237 and 402-403, positions. Predicted glycosylation sites affected the structure and stability of NA protein.

It is concluded that continuous epidemiological and virological surveillance of live bird poultry markets may help scientists to develop an effective control and preventive measures for AI viruses.

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